OTA induces intestinal epithelial barrier dysfunction and tight junction disruption in IPEC-J2 cells through ROS/Ca2+-mediated MLCK activation

Abstract

Ochratoxin A (OTA) is a frequent contaminant of feed and food worldwide. The toxicity of OTA on intestinal barrier was investigated in porcine intestinal epithelial cells (IPEC-J2). We observed that OTA induced intestinal barrier dysfunction as indicated by the reduction in transepithelial electrical resistance (TEER) and elevation in paracellular permeability to 4 kDa dextran. The barrier dysfunction was accompanied with tight junction disruption including a down-regulation in ZO-1 expression and redistribution of Occludin and ZO-1. Moreover, OTA exposure increased reactive oxygen species (ROS) generation, elevated the intracellular calcium level ([Ca²⁺]_c) and activated myosin light chain kinase (MLCK). Simultaneously, NAC, a ROS scavenger, blocked OTA-induced ROS generation, [Ca²⁺]_c elevation, barrier dysfunction and tight junction disruption, suggesting that OTA-induced ROS generation may act as a trigger. Next, we found that OTA-induced MLCK activation was inhibited by BAPTA-AM, the cytosolic Ca²⁺ chelator, demonstrating that OTA-induced MLCK activation is dependent on [Ca²⁺]_c elevation. Furthermore, inhibition of MLCK with ML-7 or inhibition of [Ca²⁺]_c elevation with BAPTA-AM markedly prevented OTA-induced barrier dysfunction and tight junction disruption. Taken together, our results indicated that OTA induces ROS generation, and then elevates the [Ca²⁺]_c and MLCK activity in turn, which finally induces barrier dysfunction and disrupts tight junction in IPEC-J2 cell monolayers.

Keywords: Intracellular calcium level; Myosin light chain kinase; Ochratoxin A; Reactive oxygen species; Tight junction.